The invention relates to the field of novel polyketides and antibiotics and to methods to prepare them. More particularly, it concerns construction of new polyketides and to libraries of polyketides synthesized by polyketide synthases derived from the picromycin PKS and other enzymes derived from Streptomyces venezuelae. 
Polyketides represent a large family of diverse compounds ultimately synthesized from 2-carbon units through a series of Claisen-type condensations and subsequent modifications. Members of this group include antibiotics such as tetracyclines, anticancer agents such as daunomycin, and immunosuppressants such as FK506 and rapamycin. Polyketides occur in many types of organisms including fungi and mycelial bacteria, in particular, the actinomycetes.
The polyketides are synthesized in vivo by polyketide synthases (PKS). This group of enzymatically active proteins is considered in a different category from the fatty acid synthases which also catalyze condensation of 2-carbon units to result in, for example, fatty acids and prostaglandins. Two major types of PKS are known which are vastly different in their construction and mode of synthesis. These are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II, xe2x80x9caromatic.xe2x80x9d
The PKS scaffold that is one subject of the present invention is a member of the group designated Type I or xe2x80x9cmodularxe2x80x9d PKS. In this type, a set of separate active sites exists for each step of carbon chain assembly and modification, but the individual proteins contain a multiplicity of such separate active sites. There may be only one multifunctional protein of this type, such as the xe2x80x9cfungalxe2x80x9d type required for the biosynthesis of 6-methyl salicylic acid (Beck, J. et al., Eur J Biochem (1990) 192:487-498; Davis, R. et al., Abstracts of Genetics of Industrial Microorganism Meeting, Montreal, Abstract P288 (1994)). More commonly, and in bacterial-derived Type I PKS assemblies, there are several such multifunctional proteins assembled to result in the end product polyketide. (Cortes, J. et al., Nature (1990) 348:176; Donadio, S. et al., Science (1991) 252:675; MacNeil, D. J. et al., Gene (1992) 115:119.)
A number of modular PKS genes have been cloned. U.S. Pat. No. 5,252,474 describes cloning of genes encoding the synthase for avermectin; U.S. Pat. No. 5,098,837 describes the cloning of genes encoding the synthase for spiramycin; European application 791,655 and European application 791,656 describe the genes encoding the synthases for tylosin and platenolide respectively.
The PKS for erythromycin, used as an illustrative system is a modular PKS. Erythromycin was originally isolated from S. erythraeus (since reclassified as Saccharopolyspora erythrea) which was found in a soil sample from the Philippine archipelago. Cloning the genes was described by Donadio, S. et al., Science (1991) 252:675. The particulars have been reviewed by Perun, T. J. in Drug Action and Drug Resistance in Bacteria, Vol. 1, S. Mitsuhashi (ed.) University Park Press, Baltimore, 1977. The antibiotic occurs in various glycosylated forms, designated A, B and C during various stages of fermentation. The entire erythromycin biosynthetic gene cluster from S. erythraeus has been mapped and sequenced by Donadio et al. in Industrial Microorganisms: Basic and Applied Molecular Genetics (1993) R. H. Baltz, G. D. Hegeman, and P. L. Skatrud (eds.) (Amer Soc Microbiol) and the entire PKS is an assembly of three such multifunctional proteins usually designated DEBS-1, DEBS-2, and DEBS-3, encoded by three separate genes.
Expression of the genes encoding the PKS complex may not be sufficient to permit the production by the synthase enzymes of polyketides when the genes are transformed into host cells that do not have the required auxiliary phosphopantetheinyl transferase enzymes which posttranslationally modify the ACP domains of the PKS. Genes encoding some of these transferases are described in WO97/13845. In addition, enzymes that mediate glycosylation of the polyketides synthesized are described in WO97/23630. U.S. Ser. No. 08/989,332 filed Dec. 11, 1997 describes the production of polyketides in hosts that normally do not produce them by supplying appropriate phosphopantetheinyl transferase expression systems. The contents of this application are incorporated herein by reference.
There have been attempts to alter the polyketide synthase pathway of modular PKS clusters. For example, European application 238,323 describes a process for enhancing production of polyketides by introducing a rate-limiting synthase gene and U.S. Pat. No. 5,514,544 describes use of an activator protein for the synthase in order to enhance production. U.S. Pat. Nos. 4,874,748 and 5,149,639 describe shuttle vectors that are useful in cloning modular PKS genes in general. Methods of introducing an altered gene into a microorganism chromosome are described in WO93/13663. Modification of the loading module for the DEBS-1 protein of the erythromycin-producing polyketide synthase to substitute the loading module for the avermectin-producing polyketide synthase in order to vary the starter unit was described by Marsden, Andrew F. A. et al. Science (1998) 279:199-202 and Oliynyk, M. et al. Chemistry and Biology (1996) 3:833-839. WO 98/01571, published Jan. 15, 1998, describes manipulation of the erythromycin PKS and polyketides resulting from such manipulation. In addition, WO 98/01546, also published Jan. 15, 1998 describes a hybrid modular PKS gene for varying the nature of the starter and extender units to synthesize polyketides.
In addition, U.S. Pat. Nos. 5,063,155 and 5,168,052 describe preparation of antibiotics using modular PKS systems.
Type II PKS, in contrast to modular PKS, include several proteins, each of which is simpler than those found in Type I polyketide synthases. The active sites in these enzymes are used iteratively so that the proteins themselves are generally monofunctional or bifunctional. For example, the aromatic PKS complexes derived from Streptomyces have so far been found to contain three proteins encoded in three open reading frames. One protein provides ketosynthase (KS) and acyltransferase (AT) activities, a second provides a chain length determining factor (CLDF) and a third is an acyl carrier protein (ACP).
The present invention is concerned with PKS systems derived from the modular PKS gene clusters which results in the production of narbomycin in Streptomyces narbonensis and of picromycin in S. venezuelae. Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosamino transferase. In S. venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase. Thus, as in the case of other macrolide antibiotics, the macrolide product of the PKS is further modified by hydroxylation and glycosylation. The nature of these clusters and their manipulation are further described below.
The invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by PKS systems derived from picromycin by using this system as a scaffold or by inserting portions of the picromycin PKS into other PKS scaffolds, and by providing recombinant forms of enzymes that further modify the resulting macrolides. Further, recombinant hosts that are modified to provide only certain activities involved in producing the endogenous antibiotic are described. Generally, many members of these libraries may themselves be novel compounds, and the invention further includes novel polyketide members of these libraries. The invention methods may thus be directed to the preparation of an individual polyketide. The individual polyketide may or may not be novel; in any case the invention provides a more convenient method of preparing it. The resulting polyketides may be further modified to convert them to antibiotics, typically through hydroxylation and/or glycosylation. Modified macrolides that are useful intermediates in the preparation of synthetic antibiotics are of particular interest. The invention also includes methods to recover novel polyketides with desired binding activities by screening the libraries of the invention.
The invention provides for the first time, the complete PKS gene cluster which ultimately results, in S. venezuelae, in the production of picromycin. The ketolide product of this PKS is narbonolide which is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin. The enzymes responsible for the glycosylation and hydroxylation are also provided.
Thus, in one aspect, the invention is directed to recombinant materials useful in the production of ketolides and their corresponding antibiotics which contain nucleotide sequences encoding at least one activity, or at least one module, or at least one protein encoded by an open reading frame of the picromycin PKS. The invention is directed also to recombinant materials useful for conversion of ketolides to antibiotics which comprise nucleotide sequences encoding the 12-hydroxylase (the picK gene) and the glycosylation enzyme which provides a glycoside residue at position 5 which enzyme is present in S. narbonensis and S. venezuelae. This aspect also provides methods to obtain the corresponding proteins, ketolides and antibiotics.
These materials are also useful as scaffolds and auxiliary reagents in preparing individual polyketides and combinatorial libraries thereof.
Thus, in another aspect, the invention is directed to a method to prepare a nucleic acid which contains a nucleotide sequence encoding a modified polyketide synthase which method comprises using the picromycin PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, or replacement. The thus modified picromycin PKS encoding nucleotide sequence can then be used to modify a suitable host cell and the cell thus modified employed to produce a polyketide different from that produced by the picromycin PKS. In addition, portions of the picromycin PKS can be inserted into other host scaffolds to modify the products thereof. Portions of the picromycin PKS can be hybridized to portions of other PKS-encoding nucleotide sequences to obtain novel nucleotide sequences with one or more reading frames encoding additional PKS alternatives. The picromycin PKS can itself be manipulated, for example, by fusing two or more of its open reading frames in order to make more efficient the production of the intended macrolide.
In another aspect, the invention relates to conversions effected by the product of the pick gene and by the product of the gene encoding glycosylation enzymes for narbonolide. The invention is also directed to polyketides thus produced and the antibiotics to which they may then be converted.
In another aspect, the invention is directed to a multiplicity of cell colonies comprising a library of colonies wherein each colony of the library contains an expression vector for the production of a different modular PKS, but derived from picromycin PKS. By xe2x80x9cderived fromxe2x80x9d picromycin PKS means simply that at least a portion of the modular PKS is identical to that found in the PKS which results the production of narbonolide and is recognizable as such. The derived portion may, of course, be prepared synthetically as well as prepared directly from DNA that originates in organisms which natively produce narbonolide. In a preferred embodiment, PKS derived from the picromycin PKS is used as a scaffold. The library of different modular PKS is in this case obtained by modifying one or more of the regions of the picromycin PKS gene cluster encoding an enzymatic activity so as to alter that activity, leaving intact the scaffold portions of picromycin PKS gene. If desired, an additional scaffold source may be used creating a hybrid scaffold. In another aspect, the invention is directed to a multiplicity of cell colonies comprising a library of colonies wherein each colony of the library contains a different modular PKS derived from the PKS gene clusters as described above. The invention is also directed to methods to produce libraries of PKS complexes and to produce libraries of polyketides and their corresponding antibiotics by culturing these colonies, as well as to the polyketide and antibiotic libraries so produced. In addition, the invention is directed to methods to screen the resulting polyketide and antibiotic libraries and to novel polyketides and antibiotics contained therein.